Fructosyltransferase enzyme, method for its production and DNA encoding the enzyme

ABSTRACT

Extracellular fructosyltransferase of Acetobacter diazotrophicus was isolated and purified and its enzymatic properties were established. Cloning, sequencing and genetic manipulation of the fructosyltransferase gene so as to produce high levels of the enzyme in recombinant prokaryotic and eukaryotic cells. Both natural and recombinant fructosyltransferase of Acetobacter diazotrophicus produce fructose-containing oligosaccharides and fructans. The enzyme yields in particular high levels of fructooligosaccharides from sucrose, such as kestose and kestotetraose which can be used as natural low-calorie sweeteners.

FIELD OF THE INVENTION

The present invention is in the field of biotechnology and is specifically concerned with the isolation, the purification, the characterization and the production of a fructosyltransferase. The invention also relates to the cloning, the sequencing and the manipulation of a fructosyltransferase gene so as to allow the production of high levels of recombinant enzyme.

BACKGROUND OF THE INVENTION

Production and utilization of microbial fructans by enzymatic transformation of sucrose are an important object for the sugar, food, and other industries. Bacterial fructosyltransferases (EC 2.4.1.10) catalyze the synthesis of oligo- and/or polyfructans by transferring fructosyl moieties from sucrose-containing saccharides to acceptor molecules. Different compounds can be used as acceptors which allows the enzymatic production of nondigestible homo- and heterooligosaccharides with beneficial effects on humans and animals.

Most of the bacterial fructosyltransferases characterized so far are levansucrases (Cote, G. L. and Ahlgran, J. A., In Science and Technology of fructans. Metabolism in microorganisms, Part I: Levan and levansucrase. CRC Press, 1993). All levansucrases catalyze the transfructosylation reaction from sucrose to a variety of acceptors such as water (sucrose hydrolysis), glucose (exchange reaction), fructose (chain elongation) and sucrose (synthesis of oligosaccharides). However, differences have been noted between these enzymes pertaining to the relative efficiency of each reaction which leads to the accumulation of oligofructans of different polymerization degree.

Several bacteria and fungi have been identified to develop transfructosylation reactions from sucrose (for review see Cote, G. L. and Ahlgran, J. A., In Science and Technology of fructans. Metabolism in microorganisms, Part I: Levan and levansucrase. CRC Press, 1993). Fructosyltransferase genes have been isolated from Bacillus subtilis (European patent application EP 0117823 A1 840905), Bacillus amyloliquefaciens (Tang, L. B. et al., Gene 96, 89-93, 1990), Streptococcus mutans (Shiroza, T. et al., J. Bacteriol. 170, No. 2, 810-816, 1988), Streptococcus salivarius (Rathsam, C. et al., J. Bacteriol. 175, No. 14, 4520-4527, 1993), Zymomonas mobilis (Ki-Bang Song et al., Biochim. Biophys. Acta 1173, 320-324, 1993) and Erwinia amylovora (Geier, G. et al., Physiological and Molecular Plant Pathology 42, 387-404, 1993). Low homology is found among the deduced amino acid sequences of fructosyltransferases isolated from different bacterial genera.

The transfructosylation system of the genus Bacillus (Gram-positive bacteria) has been well characterized. The Bacillus subtilis levansucrase is an inducible exoenzyme which catalyzes the formation of high weight polymer without accumulation of transient oligofructans of low polymerization degree. Recombinant levansucrases of Bacillus subtilis have been obtained in genetically manipulated hosts such as bacteria (Philippe, G. J. Bacteriol. 153, No. 3, 1424-1431, 1983), yeast (Scotti, P. A. et al., Yeast 10, No. 1, 29-38, 1994) and plants (Ebskamp, M. J. M. et al., Biotechnology 12, 272-275, 1994).

Acetobacter diazotrophicus is the most recently identified species of the genus Acetobacter (Gillis et al., Int. J. Sist. Bacteriol. 39, 361-364, 1989). The cells are Gram-negative, N₂ -fixing, acid-tolerant, microaerobic, straight rods with rounded ends, about 0.7 to 0.9 by ±2 μm, motile by lateral or peritrichous flagella. The bacteria are non-pathogenic and further distinguished by their ability to establish beneficial association with sugarcane. However, the molecular biology of the bacterium has been poorly investigated.

SUMMARY OF THE INVENTION

Acetobacter diazotrophicus secretes a constitutive fructosyltransferase with levansucrase activity. The enzyme has utility for the production of fructose-containing oligosaccharides and levan. Fructose polymers have two characteristic properties, their nondigestibility and selective utilization by beneficial intestinal bacteria, which make them useful as low-calorie dietary fiber for relief of constipation, improvement of blood lipid composition, cholesterol reduction, and suppression of intestinal putrefactive substances. During the course of sucrose transformation the Acetobacter diazotrophicus fructosyltransferase accumulates a high yield of fructooligosaccharides, particularly kestose and kestotetraose which can serve as natural low-calorie sweeteners. Levan can also be used as a source of fructose, a blood plasma volume extender, an emulsifier, an encapsulating agent, etc.

Given the importance of fructooligosaccharides and levan as foodstuffs as well as their industrial applications, it is an object of the invention to produce the fructosyltransferase of Acetobacter diazotrophicus both by natural and by recombinant procedures.

The invention provides a method for isolating, characterizing and producing an Acetobacter diazotrophicus fructosyltransferase by natural and recombinant procedures.

The fructosyltransferase of Acetobacter diazotrophicus is a constitutive exoenzyme which is accumulated in the culture medium yielding more than 70% of total secreted proteins. The culture supernatant can be used as a crude fructosyltransferase solution, or the enzyme be purified by any conventional method applicable here, preferably ion-exchange chromatography.

The fructosyltransferase of Acetobacter diazotrophicus is a levansucrase which yields a high level of oligofructans of low polymerization degree. During the course of sucrose transformation 55% of fructose transferred by the enzyme is accumulated as kestotriose and kestotretraose. These fructooligosaccharides are high quality sweeteners with many applications in the food industry. The enzyme can be efficiently applied, therefore, for producing kestose and kestotetraose from sucrose, and it is also useful in the production of high weight levan.

The invention also comprises the nucleotide sequence of the fructosyltransferase gene of Acetobacter diazotrophicus isolated from a genomic library by complementing EMS-treated mutants of A. diazotrophicus unable to produce levan.

The production of the fructosyltransferase of A. diazotrophicus by recombinant procedures allows higher levels of enzyme production as well as the synthesis of the enzymatic reaction products directly in a convenient host. Also provided for are strains from E. coli and the yeast Pichia pastoris genetically manipulated so as to produce elevated levels of the recombinant fructosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "Acetobacter" refers to a particular genus of bacteria described in detail in Bergey's Manual of Determinative Bacteriology, Buchanan and Gibbons eds., Williams and Wilkins Publishers. The specific strain of Acetobacter from which the enzyme fructosyltransferase and the corresponding gene were isolated is Acetobacter diazotrophicus SRT4 (CBS deposit number CBS 549.94, deposition date 10 Nov. 1994).

For the purpose of the present invention the term "fructosyltransferase" refers to one or more potypeptides having fructosyltransferase activity. Fructosyltransferase activity refers to the property of enzymatically transferring fructosyl moieties from sucrose-containing saccharides to acceptor molecules to yield fructooligosaccharides and fructans.

The term "recombinant protein" as used herein intends to cover a polypeptide encoded by genomic, cDNA, semisynthetic or synthetic nucleic acid sequences which, by virtue of their origin or manipulation: (1) are not associated with all or a portion of the polynucleotide with which they are associated in nature or in the form of a library; and/or (2) are linked to a polynucleotide sequence other than that to which they are linked in nature. The recombinant protein displays substantially the same biological properties as the naturally occurring protein.

In a first aspect, the present invention provides an isolated and purified DNA having a nucleotide sequence which essentially corresponds to or hybridizes with a nucleotide sequence comprised in the fructosyltransferase gene, shown in Seq.Id. No.1, of the bacterium Acetobacter diazotrophicus.

The words "a nucleotide sequence comprised in the fructosyltransferase gene" are intended to include several possibilities. One option is a DNA which covers the complete fructosyltransferase gene, i.e. the gene inclusive of regulatory and coding sequences. Another option is a DNA essentially consisting of the coding sequence, or a part of the coding sequence, and lacking the fructosyltransferase promoter region. One such possibility is a fructosyltransferase cDNA obtained by reverse transcription from fructosyltransferase mRNA. Furthermore, the invention relates to oligonucleotides of sufficient length to be specific for fructosyltransferase DNA or RNA. Such oligonucleotides may be useful as fructosyltransferase-specific probes or primers and will usually have a length of from about 7 or 8, preferably from about 9 or 10 nucleotides up to about 40 or 50 nucleotides, preferably to about 25 or 30 nucleotides, or longer, up to the full length of the gene.

The words "a nucleotide sequence which essentially corresponds to or hybridizes with" are intended to include single stranded and double stranded DNA. In the case of single stranded DNA, both the corresponding and the complementary sequence are intended to be included by this wording.

The correspondence need not be 100%, i.e. DNA having a certain homology to the sequence shown in Seq.Id.No.1 is intended to be included. DNA which has a homology of at least 60%, preferably a homology of at least 70% to said sequence is intended to be covered. It is especially intended to cover DNA having the same function and/or effect, even though the sequence differs from the one shown in Seq.Id.No.1. Thus, the invention is intended to include any changes in the fructosyltransferase coding region which either lead to the same amino acid sequence or to an amino acid sequence which, notwithstanding one or more deviations from the original amino acid sequence, corresponds to an enzyme having essentially fructosyltransferase activity.

For example, the invention intends to cover not only the genomic DNA and cDNA which codes for the fructosyltransferase enzyme of an Acetobacter diazotrophicus, but also DNA coding for related fructosyltransferase enzymes, e.g. DNA coding for the fructosyltransferase enzyme of related bacteria. Fructosyltransferase enzymes are considered to be related when they have a similar fructosyltransferase activity, and/or are recognized by the same anti-fructosyltransferase antibody, and/or are encoded by DNA having a homology of at least 60 or 70%.

According to a preferred embodiment of the invention, the DNA comprises a nucleotide sequence coding for an enzyme having fructosyltransferase activity. More preferably, said nucleotide sequence codes for an Acetobacter diazotrophicus fructosyltransferase enzyme having the amino acid sequence shown in Seq.Id. No.2. More specifically, said nucleotide sequence essentially consists of the nucleotide sequence shown in Seq.Id.No.1.

A further aspect of the present invention provides a recombinant polynucleotide comprising a DNA as defined above (a fructosyltransferase-specific DNA) and a nucleotide sequence of a cloning or expression vector, wherein said polynucleotide is able to direct the expression of a protein in a suitable host, more in particular a protein with fructosyltransferase activity. Said host may be a bacterium, such as a strain of E. coli, in which case the plasmid pUCLS20 is a good example of a suitable recombinant polynucleotide. The host may also be a yeast, such as the yeast Pichia pastoris, in which case the plasmid pPSLS20 is a good example of a suitable recombinant polynucleotide. The invention is not limited to particular kinds of hosts, however. In principle, any prokaryotic or eukaryotic cell may be used.

A further aspect of this invention is a host cell transformed with a recombinant polynucleotide as defined above. As indicated above, the host cell may for example be a bacterium, such as a strain of E. coli, e.g. the strain Lev1 of E. coli, or a yeast, such as the yeast Pichia pastoris, e.g. the strain Lev2 of Pichia pastoris.

An important further aspect of this invention is a proteinaceous substance having fructosyltransferase activity which comprises a polypeptide having an amino acid sequence essentially as shown in Seq.Id.No.2, or a fragment of the same. In a particularly preferred embodiment of the present invention, the proteinaceous substance having fructosyltransferase activity has a molecular weight of about 60000 daltons and an isoelectric point of about 5.5, is stable in a pH range between 4 and 9 and in a temperature range between 10° and 70° C., is active in the presence of 2% SDS, has an activity recovered after 5M urea treatments, has a specific activity of 2600 U/mg, has a Km for sucrose hydrolysis at 30° C. and pH 5.8 of about 12 mM, is not able to transfer fructosyl moieties to inulin and has a low levanase activity.

The invention covers the enzyme irrespective of how it has been produced, for example by recombinant DNA/genetic engineering technology, chemical synthesis, enzymatic degradation, or a combination thereof. Further, the invention not only covers the enzyme as such, but also in the form of a fusion protein or as a protein physically or chemically bound to any substance and having fructosyltransferase activity.

Another aspect of this invention is a method for producing a proteinaceous substance having fructosyltransferase activity, comprising the expression in a suitable host of a DNA as defined herein which codes for a fructosyltransferase enzyme. As stated above, the expression may be in prokaryotic or eukaryotic cells. This invention includes a method as defined above wherein the fructosyltransferase enzyme produced is recovered from the cells and/or the culture medium.

The invention furthermore provides a method for producing a proteinaceous substance having fructosyltransferase activity, comprising culturing a strain of Acetobacter diazotrophicus and recovering the fructosyltransferase enzyme produced. Preferably the Acetobacter diazotrophicus strain SRT4 (CBS 549.94) is used. Again the fructosyltransferase enzyme produced may be recovered, preferably from the culture medium. Recovery may be in the form of the culture medium as such, or by isolation and purification of the enzyme from the culture medium.

The isolated fructosyltransferase is characterized by the following enzymatic properties.

(1) The enzyme acts at least on sucrose and raffinose to transfer the fructosyl group to a broad range of acceptor (or receptor) molecules.

(2) The enzyme hardly exhibits activity on kestose and nistose.

(3) The enzyme shows low levanase activity and does not transfer fructosyl moieties to inulin.

(4) The enzyme has optimal activity at pH 5 and is stable in the range from 4 to 9.

(5) The enzyme is active in a temperature range from 10° to 70° C.

(6) The enzyme activity is not affected in the presence of 2% SDS.

(7) The enzyme consists of a single polypeptide of 60000 daltons as estimated by SDS-polyacrylamide gel electrophoresis.

(8) The isoelectric point of the enzyme is 5.5.

(9) The enzyme is susceptible to the inhibitive effect of the ions of mercury.

(10) The Km value for sucrose hydrolysis at 30° C. and pH 5.8 is 11.8 mM.

(11) The specific activity of the enzyme is 2600 U/mg. One unit of enzyme is defined as the amount of enzyme releasing 1 μmole of glucose per minute at 42° C. and pH 5.2.

The fructosyltransferase used in the inventive method can be naturally produced by inoculating a suitable culture medium with the strain SRT4 of Acetobacter diazotrophicus and conducting culturing of the microorganism under shaking or aeration at a temperature of 20° C. to 40° C., preferably 30° C. and at a pH value of 5 to 6.5, preferable 5.5 for a length of time of 12 hours to 3 days. In the culture medium preferable carbon sources include glucose, fructose, sucrose, glycerol, sorbitol and mannitol. Generally satisfactory results can be obtained by using a culture medium containing 2% of mannitol, 0.1% of yeast extract, 0.1% of tryptone, 0.12% of KH₂ PO₄, 0.04% of K₂ HPO₄, 0.02% of MgSO₄.7H₂ O, 0.002% of CaCl₂.H₂ O, 0.001% of FeCl₃ and 0.0002% of Na₂ MoO₄ having a pH of 6.0.

The naturally occurring fructosyltransferase of Acetobacter diazotrophicus is accumulated in the culture medium during the late phase of bacterial growth representing more than 70% of the total extracellular proteins. Since it is not inducible with sucrose, the enzyme can be obtained by culturing the microorganism in the presence of a preferable carbon source other than sucrose so that levan is not formed in the culture medium, which gives a great practical advantage in the recovery of the secreted enzyme.

After culturing, the cells are removed, preferably by centrifugation, and the supernatant can be used as a crude enzyme solution or if necessary the enzyme can be purified according to a conventional enzymological procedure, preferably ion-exchange chromatography.

The invention also provides the polynucleotide sequence coding for the fructosyltransferase of Acetobacter diazotrophicus. This DNA sequence reveals low homology with known fructosyltransferase genes. Recombinant DNA molecules comprising the provided coding sequence can be constructed by genetic engineering techniques to express desired levels of the enzyme in a variety of host cells.

Of particular interest is the expression of fructosyltransferase in a recombinant host so as to synthesize the transfructosylation products directly in the said host. Cells of interest for fructosyltransferase expression may be eukaryotic or prokaryotic, being preferred eukaryotic cells such as yeast and plant cells. Of interest is also the construction of recombinant DNA molecules comprising a modified fructosyltransferase gene so as to modulate the catalytic properties of the recombinant enzyme.

The invention further provides recombinant DNA molecules so as to produce elevated levels of the recombinant fructosyltransferase in genetically manipulated cells of E. coli and the yeast Pichia pastoris. Increased levels of expression may be achieved by a variety of genetic manipulations, including placing the encoded gene on multi-copy plasmids, and/or operably linking high level promoters and other transcriptional control sequences (operators, attenuators and the like). Thus the regulation of the fructosyltransferase gene may be altered so as to provide for inducible or constitutive expression in recombinant host cells.

The invention also provides a method for producing fructooligosaccharides and/or fructans by means of sucrose transfructosylation comprising contacting sucrose under suitable transfructosylation conditions with a fructosyltransferase enzyme or with cells producing a fructosyltransferase enzyme and optionally recovering the fructooligosaccharides produced, wherein said fructosyltransferase enzyme is a proteinaceous substance as defined herein or a proteinaceous substance obtained by a method as defined herein. Additionally, the fructosyltransferase enzyme or cells producing fructosyltransferase enzyme may be used in an immobilized form, physically or chemically coupled to a carrier material.

The fructosyltransferase of Acetobacter diazotrophicus can be used to transfer fructosyl residues to different acceptor compounds, which allows for example the synthesis of homo- and heterooligosaccharides or fructans, the hydrolysis of sucrose, etc. The enzyme can be used also to eliminate fructose from any substance that can function as donor of such residue. Using this enzyme it is possible to change the properties of an organism by changing its carbohydrate profile through the fructosyltransferase expression. As applications, for example, the products of the transfructosylation reaction can be obtained directly from said organism or alternatively the presence of said products, at low levels, could change the form of the crystals of a substance obtained from said organism, so as to facilitate its purification.

STRAIN DEPOSITS

An Acetobacter diazotrophicus strain SRT-4 was deposited on 10 Nov. 1994 with the Centraalbureau voor Schimmelcultures (CBS), The Netherlands, under the provisions of the Budapest Treaty and received accession number CBS 549.94.

An E. coli strain S17-1 containing p21R1 was deposited on 10 Nov. 1994 with the Centraalbureau voor Schimmelcultures (CBS), The Netherlands, under the provisions of the Budapest Treaty and received accession number CBS 550.94.

EXAMPLES

Examples are offered by way of illustration, not by way of limitation.

Example 1

Isolation of a levan-producing strain of Acetobacter diazotrophicus.

Stem segments (1 cm size) of sugarcane cultivar Ja60-5 were collected, superficially sterilized and inoculated into vials with 3 mL of semisolid LGI medium consisted of: K₂ HPO₄ 0.02%, KH₂ PO₄ 0.06%, MgSO₄.7H₂ O 0.02%, CaCl₂.2H₂ O 0.002%, Na₂ MoO₄.2H₂ O 0.0002%, FeCl₃ 0.001%, bromothymol blue 0.0025%, sucrose 10%, agar 0.18%, final pH 6.0. Semisolid LGI medium was reported for selective isolation of nitrogen-fixing bacteria of the species Acetobacter diazotrophicus (Cavalcante V. A. et al., Plant Soil 108: 23-31, 1988). Vials were incubated at 30° C. until growth was observed, such vials were replicated into semisolid LGI medium and those showing the typical orange-yellow surface growth were streaked out on LGI medium plates. Several mucous colonies were taxonomically characterized and classified as Acetobacter diazotrophicus, one of the isolates was named strain SRT4.

Example 2

Analysis of the extracellular polymer produced by Acetobacter diazotrophicus strain SRT4 grown on sucrose-containing media.

Bacteria were grown on solid LGI medium at 30° C. for 7 days. The extracellular polymer synthesized was collected from the medium surface with distilled water. After bacteria were removed by centrifugation, the polymer was precipitated with 2 volumes of ethanol, redissolved in distilled water, treated with saturated phenol, twice precipitated with ethanol, dialyzed against distilled water and freeze-dried. The molecular weight of the purified polymer was 7×10⁶ daltons, as estimated by gel filtration on a Sephacryl S-500 column (20×0.8 cm) eluted with 0.2M NaCl at a flow rate of 12 ml h⁻¹. Total acid hydrolysis of purified polymer was performed with 0.5M H₂ SO₄ at 100° C. for 15 min and neutralized with Ba(OH)₂. After total hydrolysis, the polymer composition was found to be fructose, as analyzed by HPLC using a Nucleosil NH₂ column (25×0.8 cm) eluted with 80% acetonitrile in water at a flow rate of 0.4 ml min⁻¹ in an isocratic way. Polymer analysis by ¹³ C-NMR spectroscopy revealed the presence of β-D-(2,6)-linked fructofuranosyl residues in a greater extent and a low rate of β-D (2-1)-linkages. This polymer structure is consistent with bacterial levans.

Example 3

Production of the exoenzyme fructosyltransferase of Acetobacter diazotrophicus.

The fructosyltransferase of Acetobacter diazotrophicus is a constitutive exoenzyme which is accumulated in the culture medium during the late phase of growth yielding more than 70% of total extracellular proteins.

The Acetobacter diazotrophicus strain SRT4 was grown in a 5 L fermenter (3.5 L working volume) B. E Marubishi (Tokyo, Japan) to stationary phase in a medium consisting of: mannitol 2%, yeast extract 0.1%, tryptone 0.1%, KH₂ PO₄ 0.12%, K₂ HPO₄ 0.04%, MgSO₄. 7H₂ O 0.02%, CaCl₂.H₂ O 0.002%, FeCl₃ 0.001%, Na₂ MoO₄ 0.0002%. The fermentation conditions were: temperature 30° C., pH 5.5, stirring speed 400 rpm, aeration 1 vvm (1 volume per minute). The culture reached a stationary OD₆₂₀ of 9.5 after fermentation for 60 hrs. The cells were removed by centrifugation and the culture supernatant was concentrated 5 fold using a rotatory evaporator and dialyzed against 20 mM Tris-HCl pH 7.0. Subsequently, ammonium sulfate was added to give 70% saturation. After centrifugation, the precipitate was dissolved in 20 mM Tris-HCl pH 7.0, dialyzed against the same buffer and applied to a column (2.5×13 cm) of DEAE-Sepharose CL-6B (fast flow). The proteins absorbed on the column were selectively eluted with a linear concentration gradient of NaCl in buffer 20 mM Tris-HCl pH 7.0. The fractions with fructosyltransferase activity eluted with the NaCl concentrations of 0.2 to 0.25M. The eluted fractions were pooled, dialyzed against a solution of 1% NH₄ HCO₃ pH 8.0 and lyophilyzed. The purified enzyme was applied on SDS-PAGE showing a single band of approximately 60 000 daltons. The N-terminal sequence of the mature fructosyltransferase is Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu (Seq.Id.No.3) as determined by the Edman degradation procedure.

The following table shows the fructosyltransferase activity, the protein content, the specific fructosyltransferase activity and the yield in the fructosyltransferase-active fractions obtained in the individual steps of the purification procedure described above.

    ______________________________________                                                     Total   Total   Specific                                                                              Reco- Purifi-                               Purification                                                                               activity                                                                               protein activity                                                                              very  cation                                step        (U)     (mg)    (U/mg) (%)   (fold)                                ______________________________________                                         Culture super-                                                                             449280  288     1560   100   1                                     natant (10 liters)                                                             Ammonium sulfate                                                                           323880  169     1507   57    0.96                                  (70% saturation)                                                               DEAE-Sepharose                                                                             128991  49      2618   28    1.68                                  CL-6B (fast flow)                                                              ______________________________________                                    

The isolated fructosyltransferase is characterized by the following enzymatic properties.

(1) The enzyme acts at least on sucrose and raffinose to transfer the fructosyl group to a broad range of receptor molecules.

(2) The enzyme hardly exhibits activity on kestose and nistose.

(3) The enzyme shows low levanase activity and does not transfer fructosyl moieties to inulin.

(4) The enzyme has optimal activity at pH 5 and is stable in the range from 4 to 9.

(5) The enzyme is active in a temperature range from 10° to 70° C.

(6) The enzyme activity is not affected by 2% SDS.

(7) The isoelectric point of the enzyme is 5.5.

(8) The enzyme is susceptible to the inhibitive effect of the ions of mercury.

(9) The Km value for sucrose hydrolysis at 30° C. and pH 5.8 is 11.8 mM.

(10) The specific activity of the enzyme is 2600 U/mg.

Example 4

Production of fructooligosaccharides by the action of the A. diazotrophicus fructosyltransferase on sucrose.

Levansucrase was added to a reaction mixture containing sucrose 1M in acetate buffer 0.1M, p.H 5.8, in a ratio of 30 units of enzyme per gram of sucrose. The reaction was conducted at 30° C. for 3 hours. Analysis of sugar products by paper chromatography on Whatman 3 MM paper in 4:6:3 pyridine:butan-1-ol:water revealed that 55% of total released fructose was accumulated in kestose in the reaction mixture. A further analysis of the reaction products was carried out by high performance liquid chromatography (HPLC) on Dionex carbopac TM PAL (column 4×250 mm) showed the following sugar composition: 31% glucose (G); 17% fructose (F); 23% sucrose; 18% kestose (GF2); 6% nistose (GF3); 3% fructosylnistose (GF4); 2% fructan (GF>4).

Example 5

Isolation of Acetobacter diazotrophicus mutants deficient in levan synthesis (Lev⁻ phenotype) by EMS mutagenesis.

Lev⁻ mutants were obtained by mutagenesis with ethylmethanesulfonate (EMS) according to the method described by Miller (Miller, J. H. 1972, Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor. N.Y.) with some modifications. The A. diazotrophicus strain SRT4 was grown aerobically in SB medium for 48 hours at 30° C. Bacteria (1.5 ml culture in an eppendorf tube) were harvested by centrifugation, washed in 1.5 ml of LGI medium salts, resuspended in 0.5 ml of 2% EMS in 0.2M Tris-HCl pH 7.4 and incubated for 90 minutes at 30° C. The treated cells were collected by centrifugation, washed with 1 ml of 5% NaS₂ O₃ in 0.2M Tris-HCl pH7.4, resuspended in 3 mL of SB medium supplemented with glycerol 1% and grown with aeration for 16 hours at 30° C. The bacterial culture was plated onto LGIE medium (LGI medium salts, tryptone 0.1%, yeast extract 0.02%) with sucrose 5% and glycerol 1% and incubated at 30° C. Colonies showing a non-mucous phenotype were picked, grown in liquid LGIE medium and assayed for fructosyltransferase activity. Nine Lev⁻ mutants were obtained at a frequency of 2.5×10⁻⁴. Two classes of mutants were isolated according to the phenotype: class I mutants did not show neither extracellular nor intracellular fructosyltransferase activity, probably due to mutations in the enzyme gene; class II mutants did not show fructosyltransferase activity in the culture supernatant but still maintain the intracellular activity.

Example 6

Construction of a genomic clone coding for the A. diazotrophicus fructosyltransferase.

Routine recombinant DNA procedures were performed by standard methods essentially as described in Maniatis 1989, Molecular Cloning, CSH, N.Y., USA.

A genomic library of A. diazotrophicus strain SRT4 was constructed in the broad-host-range cosmid pPW12, a derivative of pLAFR1 (Friedman et al., Gene 18, 289-296, 1982) with a synthetic sequence containing the EcoRI-BamHI-EcoRI sites inserted at the EcoRI site (AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, East Sussex, BN19KQ, United Kingdom).

Total DNA from A. diazotrophicus strain SRT4 was partially digested with the restriction endonuclease Sau3A I. Fragments of 15-30 kb were isolated from a low melting temperature agarose gel and cloned into BamHI-cleaved and dephosphorylated vector pPW12. The library was packaged into lambda phage particles, transferred by infection to E. coli strain S17-1 (Simon, R. et al., pp 98-106, en A. Puhler ed. Molecular Genetics of the bacteria-plant interaction, Springer-Verlag, Berlin) and plated onto LB medium supplemented with tetracycline 12 ∝g/ml. Then the gene bank was collected and transferred by conjugation to a Lev⁻ class I mutant strain isolated from the Acetobacter diazotrophicus strain SRT4.

For conjugal mating the Lev⁻ mutant strain of A. diazotrophicus was grown aerobically for 36 hours at 30° C. in SB medium with 1% glycerol as carbon source, 1.5 ml of bacterial culture were sedimented by centrifugation for 5 minutes at 12000 rpm and the cells resuspended in 0.3 mL of LGI medium salts. Simultaneously 1 ml of the gene library kept in glycerol was inoculated to 2 ml of LB medium and incubated with aeration at 37° C. for 3 hours. The cells were harvested by centrifugation and resuspended in 0.3 ml of M9 medium salts. The A. diazotrophicus mutant strain and the E. coli cells carrying the gene library were mixed on GYC agar plates (5% glucose, 1% yeast extract, 3% CaCO₃) (De Ley, J et al., pp 268-274, en N. R. Krieg & J. G. Holt ed Bergey's manual of Systematic Bacteriology, 8^(th) ed. The Williams & Wilkins Co., Baltimore, 1984) and grown for 48 hours at 30° C. The mating mixture was collected and plated onto LGIE agar medium with 5% sucrose supplemented with ampicillin 25 μg/ml (included to select against E. coli) and tetracycline 20 μg/ml for transconjugant selection. Individual colonies which recovered the mucous phenotype were picked. Plasmidic DNA from these revertants was purified, used to transform the E. coli strain S17-1 and transferred back by conjugation to the A. diazotrophicus mutant strain ascertaining that the phenotypic complementation of the Lev⁻ mutant was due to the information carried on the plasmids.

As a result of restriction endonuclease mapping, two recombinant cosmids identified as p21R1 and p21R2 were found to share a common 7.8-kb region. By several subcloning and complementation experiments, the fructosyltransferase gene was located in a 2.3 kb BglII fragment. This fragment was cloned into pUC18 (C. Yanisch-Perron et al., 1985, Gene 33: 103-119) and named pUCLS23. Nucleotide sequencing of this region revealed the presence of an open reading frame which codes for a protein with certain homology to the fructosyltransferases published so far. A smaller 2.0 kb SmaI fragment from pUCLS23 containing only this open reading frame was ligated in-frame into the 5'-region of lacZ' of pUC18 vector (C. Yanisch-Perron et al., 1985, Gene 33: 103-119) under the control of the P_(lac) promoter. The constructed plasmid named pUCLS20 was used to transform the E. coli strain 71-18 (C. Yanisch-Perron et al., 1985, Gene 33: 103-119). The resulting recombinant strain identified as Lev1 was grown in LB medium at 37° C. and the expression of the cloned gene was induced with isopropylthiogalactoside (IPTG). The polypeptide expressed in E. coli was detected in western blot by rabbit antibodies raised against the A. diazotrophicus fructosyltranferase. The recombinant protein exhibited the expected molecular mass (60 000 daltons) and showed fructosyltransferase activity as the natural enzyme.

These results demonstrate that the identified open reading frame in the recombinant plasmid pUCLS23 codes for the fructosyltransferase of Acetobacter diazotrophicus. The nucleotide sequence of the fructosyltransferase gene corresponds to the Seq.Id.No.1. The amino acid sequence deduced from the nucleotide sequence of said gene corresponds to the Seq.Id.No.2.

Example 7

Production of an enzymatic preparation with fructosyltransferase activity in recombinant E. coli.

The recombinant E. coli strain Lev1 was inoculated to an erlenmeyer containing 300 ml of LB medium supplemented with 0.5% glucose and grown in a rotatory shaker at 37° C. for 16 hours. This culture was used to inoculate a 5 L fermenter (working volume 3.5 L) B. E Marubishi (Tokyo, Japan) containing LB medium supplemented with 0.5% glucose at an initial OD₅₃₀ of 0.05. The fermentation conditions were as follows: 37° C., pH 6.8-7.2, stirring speed 350 rpm and aeration 1 VVM (1 volume per minute). When the OD₅₃₀ reached a value of 2 the culture was induced with 1 mM of isopropylthiogalactoside (IPTG) and kept in the same conditions for further 6 hours. The cells collected by centrifugation were resuspended in an equal volume of buffer 0.1M sodium acetate, pH 5.8 and sonicated in a B. BRAUN, model LABSONIC 2000 apparatus for five cycles of 30 seconds each with 1 min intervals on ice. The production of the recombinant enzyme was determined immunologically in the cellular crude, representing approximately the 5% of total soluble proteins. The recombinant fructosyltransferase expressed in E. coli displayed the same catalytic properties as the natural enzyme.

Example 8

Production of an enzymatic preparation with fructosyltransferase activity in recombinant yeast Pichia pastoris.

In order to obtain extracellular expression of the recombinant fructosyltransferase in the methylotrophic yeast Pichia pastoris, the following experiments were performed.

Two oligonucleotides were synthesized: 5'-CATGGCGGCCCGCTCTTCCCC-3' (Seq.Id.No.4) and 5'-GGGGAAGAGCGGGCCGC-3' (Seq.Id.No.5) and hybridized to each other, resulting in a double stranded DNA fragment with one extremity compatible with the NcoI recognition site and one blunt-ended extremity. The sequence of the oligonucleotides was chosen such that the fragment coded for the first six amino acids of the N-terminal region of the mature fructosyltransferase. This synthetic fragment was blunt ligated to the 2.0-kb SmaI fragment of the plasmid pUCLS23 obtaining a NcoI fragment with the entire coding sequence of the mature enzyme, which was then inserted into the cleaved NcoI site of the integrative vector pPS7 (European patent application EP 438 200 A1) resulting in an "in frame" fusion of the Saccharomyces cerevisiae Suc2 signal peptide coding sequence and the fructosyltransferase gene under the control of the alcohol oxidase 1 (AOX1) methanol inducible promoter. This construction named pPSLS20 was PvuII digested and used to transform the Pichia pastoris mutant strain MP36 (his3⁻) (European patent application EP 438 200 A1) according to the procedure described by Meilhoc et at. (Meilhoc et al. (1990), Bio/Technology 8: 223-227). The transformed cells were selected on minimal medium G (P. Galzy, 1957, Paris. C. R. Acad. Sci. 245: 2423-2427) with 2% glucose as carbon source and immunologically screened for fructosyltransferase production. A recombinant yeast expressing high levels of fructosyltransferase after induction with methanol was named strain Lev2.

The production of recombinant fructosyltransferase by Pichia pastoris strain Lev2 was studied in a 5 L fermenter (3.5 L working volume) B. E Marubishi (Tokyo, Japan) in YPG medium (1% yeast extract 1%, peptone 2%, glucose 2%), inoculated at an initial OD₅₃₀ of 0.2, pH 5.2, temperature 30° C., aeration 1 VVM (1 volume per minute), stirring speed 350 rpm. At an optical density of 60 a gradually increasing flow of methanol was fed reaching 3.5 g/h/L, stirring speed was increased from 350 to 750 rpm. The culture was induced during 120 hours.

The enzymatic activity in the culture supernatant increased during induction reaching 5000 U/mL (2 g/L). Following cell disruption with glass beads (Maniatis et a1. (1989) Molecular Cloning, CSH, New York, USA) it was determined that approximately 85% of the total enzyme produced was secreted to the culture medium.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 5                                                   (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1632 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       ATGGCGCACCGTCCGGGTGTGATGCCTCGTGGCGGCCCGCTCTTCCCCGGGCGGTCGCTG60                 GCCGGGGTGCCGGGCTTCCCGCTGCCCAGCATTCATACGCAGCAGGCGTATGACCCGCAG120                TCGGACTTTACCGCCCGCTGGACACGTGCCGACGCATTGCAGATCAAGGCGCATTCGGAT180                GCGACGGTCGCGGCCGGGCAGAATTCCCTGCCGGCGCAACTGACCATGCCGAACATCCCG240                GCGGACTTCCCGGTGATCAATCCGGATGTCTGGGTCTGGGATACCTGGACCCTGATCGAC300                AAGCACGCCGATCAGTTCAGCTATAACGGCTGGGAAGTCATTTTCTGCCTGACGGCCGAC360                CCCAATGCCGGATACGGTTTCGACGACCGCCACGTGCATGCCCGCATCGGCTTCTTCTAT420                CGTCGCGCGGGTATTCCCGCCAGCCGGCGGCCGGTGAATGGCGGCTGGACCTATGGCGGC480                CATCTCTTCCCCGACGGAGCCAGCGCGCAGGTCTACGCCGGCCAGACCTACACGAACCAG540                GCGGAATGGTCCGGTTCGTCGCGTCTGATGCAGATACATGGCAATACCGTATCGGTCTTC600                TATACCGACGTGGCGTTCAACCGTGACGCCAACGCCAACAACATCACCCCGCCGCAGGCC660                ATCATCACCCAGACCCTGGGGCGGATCCACGCCGACTTCAACCATGTCTGGTTCACGGGC720                TTCACCGCCCACACGCCGCTGCTGCAGCCCGACGGCGTGCTGTATCAGAACGGTGCGCAG780                AACGAATTCTTCAATTTCCGCGATCCGTTCACCTTCGAGGACCCGAAGCATCCCGGCGTG840                AACTACATGGTGTTCGAGGGCAATACCGCGGGCCAGCGTGGCGTCGCCAACTGCACCGAG900                GCCGATCTGGGCTTCCGCCCGAACGATCCCAATGCGGAAACCCTGCAGGAAGTCCTGGAT960                AGCGGGGCCTATTACCAGAAGGCCAATATCGGCCTGGCCATCGCCACGGATTCGACCCTG1020               TCGAAATGGAAGTTCCTGTCGCCGCTGATTTCGGCCAACTGCGTCAATGACCAGACCGAA1080               CGGCCGCAGGTGTACCTCCATAACGGAAAATACTATATCTTCACCATCAGCCACCGCACG1140               ACCTTCGCGGCCGGTGTCGATGGACCGGACGGCGTCTACGGCTTCGTGGGTGACGGCATC1200               CGCAGTGACTTCCAGCCGATGAACTATGGCAGCGGCCTGACGATGGGCAATCCGACCGAC1260               CTCAACACGGCGGCAGGCACGGATTTCGATCCCAGCCCGGACCAGAACCCGCGGGCCTTC1320               CAGTCCTATTCGCACTACGTCATGCCGGGGGGACTGGTTGAATCGTTCATCGACACGGTG1380               GAAAACCGTCGCGGGGGTACCCTGGCGCCCACGGTCCGGGTGCGCATCGCCCAGAACGCG1440               TCCGCGGTCGACCTGCGGTACGGCAATGGCGGCCTGGGCGGCTATGGCGATATTCCGGCC1500               AACCGCGCGGACGTGAACATCGCCGGCTTCATCCAGGATCTGTTCGGCCAGCCCACGTCG1560               GGTCTGGCGGCGCAGGCGTCCACCAACAATGCCCAGGTGCTGGCGCAGGTTCGCCAATTC1620               CTGAACCAGTAA1632                                                               (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 543 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       MetAlaHisArgProGlyValMetProArgGlyGlyProLeuPhePro                               151015                                                                         GlyArgSerLeuAlaGlyValProGlyPheProLeuProSerIleHis                               202530                                                                         ThrGlnGlnAlaTyrAspProGlnSerAspPheThrAlaArgTrpThr                               354045                                                                         ArgAlaAspAlaLeuGlnIleLysAlaHisSerAspAlaThrValAla                               505560                                                                         AlaGlyGlnAsnSerLeuProAlaGlnLeuThrMetProAsnIlePro                               65707580                                                                       AlaAspPheProValIleAsnProAspValTrpValTrpAspThrTrp                               859095                                                                         ThrLeuIleAspLysHisAlaAspGlnPheSerTyrAsnGlyTrpGlu                               100105110                                                                      ValIlePheCysLeuThrAlaAspProAsnAlaGlyTyrGlyPheAsp                               115120125                                                                      AspArgHisValHisAlaArgIleGlyPhePheTyrArgArgAlaGly                               130135140                                                                      IleProAlaSerArgArgProValAsnGlyGlyTrpThrTyrGlyGly                               145150155160                                                                   HisLeuPheProAspGlyAlaSerAlaGlnValTyrAlaGlyGlnThr                               165170175                                                                      TyrThrAsnGlnAlaGluTrpSerGlySerSerArgLeuMetGlnIle                               180185190                                                                      HisGlyAsnThrValSerValPheTyrThrAspValAlaPheAsnArg                               195200205                                                                      AspAlaAsnAlaAsnAsnIleThrProProGlnAlaIleIleThrGln                               210215220                                                                      ThrLeuGlyArgIleHisAlaAspPheAsnHisValTrpPheThrGly                               225230235240                                                                   PheThrAlaHisThrProLeuLeuGlnProAspGlyValLeuTyrGln                               245250255                                                                      AsnGlyAlaGlnAsnGluPhePheAsnPheArgAspProPheThrPhe                               260265270                                                                      GluAspProLysHisProGlyValAsnTyrMetValPheGluGlyAsn                               275280285                                                                      ThrAlaGlyGlnArgGlyValAlaAsnCysThrGluAlaAspLeuGly                               290295300                                                                      PheArgProAsnAspProAsnAlaGluThrLeuGlnGluValLeuAsp                               305310315320                                                                   SerGlyAlaTyrTyrGlnLysAlaAsnIleGlyLeuAlaIleAlaThr                               325330335                                                                      AspSerThrLeuSerLysTrpLysPheLeuSerProLeuIleSerAla                               340345350                                                                      AsnCysValAsnAspGlnThrGluArgProGlnValTyrLeuHisAsn                               355360365                                                                      GlyLysTyrTyrIlePheThrIleSerHisArgThrThrPheAlaAla                               370375380                                                                      GlyValAspGlyProAspGlyValTyrGlyPheValGlyAspGlyIle                               385390395400                                                                   ArgSerAspPheGlnProMetAsnTyrGlySerGlyLeuThrMetGly                               405410415                                                                      AsnProThrAspLeuAsnThrAlaAlaGlyThrAspPheAspProSer                               420425430                                                                      ProAspGlnAsnProArgAlaPheGlnSerTyrSerHisTyrValMet                               435440445                                                                      ProGlyGlyLeuValGluSerPheIleAspThrValGluAsnArgArg                               450455460                                                                      GlyGlyThrLeuAlaProThrValArgValArgIleAlaGlnAsnAla                               465470475480                                                                   SerAlaValAspLeuArgTyrGlyAsnGlyGlyLeuGlyGlyTyrGly                               485490495                                                                      AspIleProAlaAsnArgAlaAspValAsnIleAlaGlyPheIleGln                               500505510                                                                      AspLeuPheGlyGlnProThrSerGlyLeuAlaAlaGlnAlaSerThr                               515520525                                                                      AsnAsnAlaGlnValLeuAlaGlnValArgGlnPheLeuAsnGln                                  530535540                                                                      (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       GlyGlyProLeuPheProGlyArgSerLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       CATGGCGGCCCGCTCTTCCCC21                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       GGGGAAGAGCGGGCCGC17                                                            __________________________________________________________________________ 

We claim:
 1. An isolated and purified DNA molecule having a nucleotide sequence which comprises the fructosyltransferase gene, shown in Seq.Id.No.1, of the bacterium Acetobacter diazotrophicus.
 2. An isolated and purified DNA molecule which comprises a nucleotide sequence coding for an Acetobacter diazotrophicus fructosyltransferase enzyme having the amino acid sequence shown in Seq. Id. No.
 2. 3. A recombinant polynucleotide comprising the DNA molecule according to claim 1 and a nucleotide sequence of a cloning or expression vector, wherein said polynucleotide is able to direct the expression of the encoded fructosyltransferase in a suitable host.
 4. A recombinant polynucleotide according to claim 3 which is able to direct expression of a protein with fructosyltransferase activity in a suitable host.
 5. A recombinant polynucleotide according to claim 3 which is able to direct expression of a protein with fructosyltransferase activity in a bacterium.
 6. A recombinant polynucleotide according to claim 3 which is able to direct expression of a protein with fructosyltransferase activity in E. coli.
 7. A recombinant polynucleotide according to claim 6 which is the plasmid pUCLS20.
 8. A recombinant polynucleotide according to claim 3 which is able to direct expression of a protein with fructosyltransferase activity in a yeast.
 9. A recombinant polynucleotide according to claim 3 which is able to direct expression of a protein with fructosyltransferase activity in the yeast Pichia pastoris.
 10. A recombinant polynucleotide according to claim 9 which is the plasmid pPSLS20.
 11. A host cell transformed with the recombinant polynucleotide of claim
 3. 12. A host cell according to claim 11 which is a bacterium.
 13. A host cell according to claim 11 which is E. coli.
 14. A host cell according to claim 11 which is the strain Lev1 of E. coli.
 15. A host cell according to claim 11 which is a yeast.
 16. A host cell according to claim 11 which is the yeast Pichia pastoris.
 17. A host cell according to claim 11 which is the strain Lev2 of Pichia pastoris.
 18. A method for producing a proteinaceous substance having fructosyltransferase activity, comprising the expression in a suitable host of a DNA molecule according to claim 1 coding for a fructosyltransferase enzyme.
 19. A method according to claim 18 wherein said expression is in prokaryotic or eukaryotic cells.
 20. A method according to claim 18 wherein the fructosyltransferase enzyme produced is recovered from the cells and/or the culture medium. 